In biomedicine, 3D fluorescence imaging with high lateral and axial resolution inside extended biological samples is desired. Conventional spatial sampling techniques such as epi-fluorescence, point scanning and sheet microscopy face inherent limiting trade-offs between spatial resolution, field-of-view, photodamage, and recording speed.
FIG. 1A is schematic drawing of epi-fluorescence illumination used in a conventional epi-fluorescence microscope. Conventional epi-fluorescence microscopy suffers from poor axial resolution (i.e. resolution in z-direction), and out-of-focus fluorescence.
Conventional selective plane illumination microscopy (SPIM) scans a thin sheet of laser light across a sample as shown in FIG. 1B. Due to the laws of diffraction and Gaussian beam propagation, conventional SPIM is limited by a tradeoff between sheet thickness (which affects axial resolution of the sample in the z-direction) and lateral extent (which affects lateral field of view: FOVx) as described by the graph shown in FIG. 1C. Moreover, SPIM suffers from prominent image artifacts such as striping and shadowing. Some examples of conventional SPIM can be found in Siedentopf, H. and Zsigmondy, R., “Über Sichtbarmachung and Gröβenbestimmung ultramikoskopischer Teilchen, mit besonderer Anwendung auf Goldrubingläser,” Annalen der Physik 315, pp. 1-39 (1902), Huisken, J., Swoger, J., del Bene, F., Wittbrodt, J. and Stelzer, E. H. K., “Optical sectioning deep inside live embryos by selective plane illumination microscopy,” Science 305, pp. 1007-1009 (2004), Dodt, H.-U. et al., “Ultramicroscopy: three-dimensional visualization of neuronal networks in the whole mouse brain,” Nat Meth 4, pp. 331-336 (2007), Keller, P. J., Schmidt, A. D., Wittbrodt, J. and Stelzer, E. H. K., “Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy,” Science 322, pp. 1065-1069 (2008), Huisken, J. and Stainier, D. Y. R., “Selective plane illumination microscopy techniques in developmental biology,” Development 136, pp. 1963-1975 (2009), Mertz, J., “Optical sectioning microscopy with planar or structured illumination,” Nat Meth 8, 811-819 (2011), Palero, J., Santos, S. I. C. O., Artigas, D. and Loza-Alvarez, P. A, “Simple scanless two photon fluorescence microscope using selective plane illumination,” Opt Express 18, pp. 8491-8498 (2010), Truong, T. V., Supatto, W., Koos, D. S., Choi, J. M. and Fraser, S. E., “Deep and fast live imaging with two-photon scanned light-sheet microscopy,” Nat Meth 8, pp. 757-760 (2011), Huisken, J. and Stainier, D. Y. R., “Even fluorescence excitation by multidirectional selective plane illumination microscopy (mSPIM),” Opt Lett 32, pp. 2608-2610 (2007), Krzic, U., Gunther, S., Saunders, T. E., Streichan, S. J. and Hufnagel, L., “Multiview light-sheet microscope for rapid in toto imaging,” Nat Meth (2012), Tomer, R., Khairy, K., Amat, F. and Keller, P. J., “Quantitative high-speed imaging of entire developing embryos with simultaneous multiview light-sheet microscopy,” Nat Meth (2012), which are hereby incorporated by reference in their entirety.
With imaging techniques that use non-diffracting self-healing Bessel beams, much of the optical power resides in side lobes of the beam, which can result in excitation of unwanted out-of-focus photons. These side lobes present an inherent trade-off of photodamage (e.g., photobleaching) that may result from this unwanted excitation, which in turn limits the useful extent of the Bessel beam illumination. Bessel beam based sheet microscopy has thus far only been demonstrated for short sheet lengths of a few tens of μm, resulting in small fields-of-view unless images are tiled at the expense of increased sample illumination and recording time. Some examples of conventional Bessel beam imaging can be found in Herman, R. M. and Wiggins, T. A., “Production and uses of diffractionless beams,” JOSA A 8, pp. 932-942 (1991), Fahrbach, F. O. and Rohrbach, A., “Propagation stability of self-reconstructing Bessel beams enables contrast-enhanced imaging in thick media,” Nat Commun 3, p. 632 (2012), Fahrbach, F. O., Simon, P. and Rohrbach, A., “Microscopy with self-reconstructing beams,” Nature Photonics 4, pp. 780-785 (2010), Planchon, T. A. et al., “Rapid three-dimensional isotropic imaging of living cells using Bessel beam plane illumination,” Nat Meth 8, pp. 417-423 (2011), Gao, L. et al., “Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens,” Cell 151, pp. 1370-1385 (2012), which are hereby incorporated by reference in their entirety.
Conventional standing-wave fluorescence imaging (SWFI) can provide wide-field fluorescence imaging by interfering two laser beams and generating a high frequency standing wave patterns as discussed in Bailey, B., Farkas, D. L., Taylor, D. L. and Lanni, F., “Enhancement of axial resolution in fluorescence microscopy by standing-wave excitation,” Nature 366, pp. 44-48 (1993), which is hereby incorporated by reference in its entirety. However, due to ambiguity along the axial dimension, SWFI is only suitable for very thin samples with sub-μm features and not suitable for thick biological tissues.